It is proposed to use (Se-injected) normal and selenium deficient (white muscle diseased, WMD) lambs to determine (1) cytochrome content of tissue fractions, (2) the involvement of selenium in formation of cytochromes, (3) the phosphorus: oxygen ratios of mitochondria (4) and calcium sequestering capacity of sarcotubular membranes. In addition, the major selenium-binding proteins from liver and glutathione peroxidase from heart will be purified and characterized from normal lambs. Cytochromes will be extracted with bile salts from tissue fractions of normal and WMD lambs and their content determined by spectrophotometric methods after preferential reduction with ascorbate or dithionite. The heme containing selenoproteins will be further characterized by spectrometric, enzymmtic, and chemical methods to determine their properties and physiological significance. These heme containing selenoproteins will be purified by the isolation method developed in our laboratory, employing ammonium sulfate fractionation, gel filtration and hydroxylapatite chromatography. Incorporation of selenium into cytochromes will be determined by isolating these compounds from noral and WMD lambs that have been injected with 75Se-selenite. If significant quantities are incorporated into a particular cytochrome, this one will be purified to determine the amount of selenium bound per mole compound. The ADP:O ratios of mitochondria from tissues of normal and WMD lambs will be determined with a polarographic oxygen electrode instrument. it is proposed to purify the major selenium binding protein from liver and glutathione peroxidase from heart by salt fractionation, chromtography on ionic and cationic resins, and by gel filtration methods. The form and amount of selenium in these proteins will be determined by ion exchange, thin-layer, and gas-liquid chromatography. Kinetic measurements will be used to ssess the pecificity of glutationine peroxidase for thiol groups and peroxy electron donors. Sarcotubular membranes will be treated with lysosomal hydrolases (lipases, phospholipases and protein hydrolases) to etermine the membrane structure necessary for proper calcium sequestering and whether lysosomal enzymes affect this process.